DESIGN AND OPTIMIZATION OF A BIOSENSOR FOR THE DETECTION AND
QUANTIFICATION OF T-LYMPHOCYTES
A Thesis
Presented to the Faculty of the Graduate School
of Cornell University
in Partial Fulfillment of the Requirements for the Degree of
Master of Engineering
by
Jennifer J. Lee
May 2005
© 2005 Jennifer J. Lee
ABSTRACT
In health care, cell enumeration by immunophenotyping has been crucial in
diagnosing and treating patients with HIV, leukemia, or bone marrow transplantation.
One key use of immunophenotyping is for detection of HIV. The increase in the
number of HIV in blood leads to a significant drop in one subset of leukocyte, the T
helper cells (CD4 cells), in the patient’s blood. Detection and quantification of CD4
cells are thus important measures for the indication of HIV infection. Many
immunophenotyping techniques have been developed in the past but the flow
cytometry has stood out as the golden standard. Flow cytometric immunophenotyping
uses the receptor proteins present on the T-helper cells, CD3, CD4, and CD45 for
quantification. This technique, however, is very costly and not affordable to all health
care sectors.
This project investigated a design and optimization of a biosensor that
quantifies the amount of T-lymphocytes in human blood. Quantification of T
lymphocytes is useful as a positive control in the case of HIV detection and as a
determinant of immunological status of the patient during immunosuppressive therapy.
A microfluidic biosensor based on liposome signal generation and amplification is
adapted to the detection of the T-lymphocytes using two specific sets of antibodies
recognizing CD3 and CD45 proteins on the surface of the cells.
This microfluidic biosensor utilized superparamagnetic beads tagged with
CD45 antibody as capture probe and sulforhodamine B entrapping immunoliposomes
tagged with CD3 antibody as reporter probe. The assay condition was optimized for
the running buffer composition and the flow time resulting in assay times of
approximately 10 minutes. Non-specific interactions of the liposomes and beads were
measured and reduced by approximately 30% using a blocking solution. The nonspecific interactions of the red blood cells were reduced by lysing the red blood cell
prior to testing. A dose response curve of varying human blood concentrations was
created to show the correlation of the signal with respect to different concentrations of
cells. The limit of detection was determined to be 80-220 T-lymphocytes.
The micro-biosensor was designed to be a simple and easy to use device that
can be manufactured and operated at low cost and was thus applicable for testing in
resource-limited settings. It has a great potential as a tool for detection and
quantification of T-lymphocytes since it uses the same biological recognition
principles as used in flow cytometry, however, its simplicity and low cost make it an
attractive alternative .